Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 2018 - 06 Augustus 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine

EC Number:

254-754-2

EC Name:

Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine

Cas Number:

40027-38-1

Molecular formula:

C21H44N2xC18H34O2

IUPAC Name:

oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine

Test material form:

solid

Details on test material:

Substance Name: N-(Oleyl alkyl)- 1,3-propanediamine mono oleate
CAS: 40027-38-1
Lot/Batch: P15-005
Appearance: Yellow paste at 20°C
Test item storage: At room temperature
Date of Production: 2015-02-18
Best before Date: 2020-02-18
Trade Name: Armolube 211
mp 30-40°C 
D = 880 kg/m³ at 40°C 
m.form. C39H78N2O2
mw 607.065
Purity: UVCB
Purity test date: 24 April 2018

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Direct plate assay
Dose-range finding test (without and with S9 (5% (v/v)); tester strains TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (reported as part of experiment 1)
First experiment (without S9 and with S9 (5% (v/v)); tester strains TA1535, TA1537 and TA98): 3.1, 6.3, 12.5, 25, 50, 250 μg/plate
Additonal experiment (without S9; Tester strain TA98): 0.78, 1.6 μg/plate

Second experiment (without S9; tester strains TA100, TA1535, TA1537, TA 98): 1.6, 3.1, 6.3, 12.5, 25 and 100 μg/plate
Second experiment (with S9 (10% (v/v)); tester strains TA100, TA1535, TA1537, TA 98): 3.1, 6.3, 12.5, 25, 50 and 250 μg/plate
Second experiment (with and without S9(10% (v/v)); tester strain WP2uvrA): 6.3, 12.5, 25, 50, 250 and 600 μg/plate

Vehicle / solvent:

- Vehicle used: Ethanol
- Justification for choice of vehicle: a previously performed solubility test showed that the test item can be dissolved in ethanol.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene; ICR-191

Remarks:

For details on positive control substances, see Table 1 and Table 2

Details on test system and experimental conditions:

Two individual experiments were performed. The dose range-finding study with tester strains TA100 and WP2uvrA was reported as part of the first experiment. The second experiment was performed to obtain more information about the possible mutagenicity of the test item in the absence and presence of 10% (v/v) S9-mix.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

METHODS: The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (1E9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in ethanol and
either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, presence of microcolonies and the reduction of the revertant colonies.
- Other: precipitation of the test item was recorded.

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 512 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 164 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 25 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 25 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 25 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 250 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 250 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Conc: 250 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRECIPITATION
- Precipitation of the test item on the plates was not observed at the start of the incubation period. Precipitation of the test item on the plates was observed at the end of the incubation period at concentrations of 1600 and 5000 μg/plate in the first experiment.
- Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period of the second experiment.

CYTOXICITY
In the first and second experiment cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester
strains in the absence and presence of S9-mix.

- In both the first and second assay, criteria for a negative response were met for all tester strains with and without metabolic activation.
- The negative and strain-specific positive control values were within the laboratory historical control data ranges except the negative control response for TA98 in the absence of S9-mix in the second experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (44 revertant colonies) when compared against relevant historical control data (41 revertant colonies), the validity of the test was considered to be not affected.

Applicant's summary and conclusion

Conclusions:

Based on the results of an Ames test, performed according to OECD guideline 471 and GLP principles, N-(Oleyl alkyl)-1,3- propanediamine mono oleate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Armolube 211 was tested in the Salmonella typhimurium (S.typhimurium; TA98, TA100, TA1535, and TA1537) and Escherichia coli (WP2uvrA) reverse gene mutation assay by direct plate incorporation method followed by an independent repeat.

Armolube 211 was a yellow paste. Ethanol was used as vehicle.

The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. Comparable cytotoxicity was observed in the absence and presence of S9-mix.

Armolube 211 did not induce a significant dose-related increase in the number of revertant colonies in each of the tester strains both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Conclusion: Armolube 211 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.